Effects of H2A.B incorporation on nucleosome structures and dynamics

Abstract

The H2A.B histone variant is an epigenetic regulator involved in transcriptional upregulation, DNA synthesis, and splicing that functions by replacing the canonical H2A histone in the nucleosome core particle. Introduction of H2A.B results in less compact nucleosome states with increased DNA unwinding and accessibility at the nucleosomal entry and exit sites. Despite being well characterized experimentally, the molecular mechanisms by which H2A.B incorporation alters nucleosome stability and dynamics remain poorly understood. To study the molecular mechanisms of H2A.B, we have performed a series of conventional and enhanced sampling molecular dynamics simulation of H2A.B- and canonical H2A-containing nucleosomes. Results of conventional simulations show that H2A.B weakens protein-protein and protein-DNA interactions at specific locations throughout the nucleosome. These weakened interactions result in significantly more DNA opening from both the entry and exit sites in enhanced sampling simulations. Furthermore, free energy profiles show that H2A.B-containing nucleosomes have significantly broader free wells and that H2A.B allows for sampling of states with increased DNA breathing, which are shown to be stable on the hundreds of nanoseconds timescale with further conventional simulations. Together, our results show the molecular mechanisms by which H2A.B creates less compacted nucleosome states as a means of increasing genetic accessibility and gene transcription.

Figure 1

Substitution of canonical H2A with H2A.B results in more open nucleosome structures (top). Sequence alignment of H2A and H2A.B histones (bottom) shows that H2A.B has a shorter docking domain (boxed), alterations to the acidic patch, and no C-terminal tails (5). Basic residues are in blue, acidic in red, hydrophobic in green, and polar in black. To see this figure in color, go online.

Figure 2

Nucleosome regions that have the most significant change in interaction energies upon H2A.B incorporation at the dimer/tetramer interface, along with their energy changes (kcal/mol). These regions correspond to 1) the H2B α₂/H4, 2) the H2A/H3-H4, and 3) the H2A docking domain/H3 interfaces. To see this figure in color, go online.

Figure 3

Stabilizing DNA-dimer contacts in H2A- and H2A.B-containing systems. Protein residues and DNA basepairs forming contacts are shown in green and yellow, respectively. Residues at the H2A/H2A.B L2 loops (see zoomed-in image on the left) have the largest difference in stabilizing contacts upon variant substitution. DNA is detached from protein core in H2A.B NCP as a result of weakened DNA-L2 loop bonds. This is in agreement with the increased distance at this interface in H2A.B NCP; see Fig. 6. Interaction energies for specific residues are in Tables 3 and 4. To see this figure in color, go online.

Figure 4

Radius of gyration for the entry and exit DNA segments in H2A and H2A.B nucleosomes during ABMD simulations. Both the entry and exit DNA segments in H2A.B systems have increased sampling and extensions relative to canonical nucleosomes, which corresponds to more DNA breathing and increased DNA exposure. For representative structures at various radii of gyration, see Figs. 7 and 8. To see this figure in color, go online.

Figure 5

Free energy landscapes of canonical- and H2A.B-containing nucleosomes. Both systems have a single energy basin; however, replacement of H2A with H2A.B results in a broader well and increased sampling of higher energy states. To see this figure in color, go online.

Figure 6

Separation distance between H2A (A and B) and H2A.B (C and D) L2 loops with their adjacent DNA basepairs in ABMD simulations. In canonical systems, these distances are stable, indicating that the L2 loops remain in contact with the DNA throughout the majority of the simulations. In H2A.B systems, there is a higher degree of detachment as the distances between the L2 loops and DNA sample a wide array of states throughout the simulations. For representative structures at various L2/DNA distances, see Figs. 7 and 8. To see this figure in color, go online.

Figure 7

Structural clusters of H2A nucleosomes extracted from ABMD simulations. Structures represent closed and semiclosed states with radii of gyration between 37.5 and 49.8 Å for entry DNA and from 39.7 to 44.3 Å for exit DNA. Distance to L2 shows the distance of the L2 loop residues to their adjacent DNA basepairs. Compared with H2A.B NCPs, the DNA L2 loop distances are lower, which is in agreement with lower radii of gyration. See Figs. S7 and S8 for side views of these clusters. To see this figure in color, go online.

Figure 8

Structural clusters of H2A.B nucleosomes extracted from ABMD simulations. Structures represent semiopen and open states with radii of gyration between 41.4 and 56.2 Å for entry DNA and from 42.1 to 48.4 Å for exit DNA. Distance to L2 shows the distance of the L2 loop residues to their adjacent DNA basepairs. Increasing radii of gyration are associated with increased DNA L2 loop distances in the open states of H2A.B NCPs. See Figs. S9 and S10 for side views of these clusters. To see this figure in color, go online.

Figure 9

Differences between atomic mutual information calculations of the H2A and H2A.B NCPs in the compact (A) and open states (B). In both cases, there are higher correlated motions in the H2A NCP. (C) The difference between the compact structure of the H2A NCP to the open H2A.B NCP state shows significant stronger correlations in the closed H2A state. To see this figure in color, go online.